Inorganic polyphosphate (polyP) is a nontoxic polymer existing in a wide range of organisms (Schröder H C, Müller W E G, eds (1999) Inorganic Polyphosphates—Biochemistry, Biology, Biotechnology. Prog Mol Subcell Biol 23:45-81; Kulaev I S, Vagabov V, Kulakovskaya T (2004) The Biochemistry of Inorganic Polyphosphates. New York: John Wiley & Sons Inc). It consists of usually linear molecules of tens to hundreds of phosphate units which are linked together via high energy phosphoanhydride bonds. Living organisms can produce this polymer metabolically at ambient temperatures, while the chemical synthesis of polyP requires high temperatures of several hundred degrees.
PolyP in Bone
Previous studies revealed that polyP molecules of different chain lengths accumulate especially in bone cells (Leyhausen G, Lorenz B, Zhu H, Geurtsen W, Bohnensack R, Müller W E G, Schröder H C. Inorganic polyphosphate in human osteoblast-like cells. J Bone Mineral Res 1998; 13:803-812; Schröder H C, Kurz L, Müller W E G, Lorenz B. Polyphosphate in bone. Biochemistry (Moscow) 2000; 65:296-303). In addition, human osteoblast-like cells contain enzymes that hydrolyze polyP, e.g. the alkaline phosphatase (ALP) (Lorenz B, Schröder H C. Mammalian intestinal alkaline phosphatase acts as highly active exopolyphosphatase. Biochim Biophys Acta 2001; 1547:254-261). PolyP is also found in platelets (Smith S A, Mutch N J, Baskar D, Rohloff P, Docampo R, Morrissey J H. Polyphosphate modulates blood coagulation and fibrinolysis. Proc Natl Acad Sci USA 2006; 103:903-908), possibly playing a role in initiation of healing of bone fractures.
PolyP has an inductive effect on osteoblasts mainly as an anabolic polymer that stimulates differentiation of bone cells and mineralization (reviewed in: Wang X H, Schröder H C, Wiens M, Ushijima H, Müller W E G. Bio-silica and bio-polyphosphate: applications in biomedicine (bone formation). Current Opinion Biotechnol 2012; 23:570-578; Wang X H, Schröder H C and Müller W E G. Enzymatically synthesized inorganic polymers as morphogenetically active bone scaffolds: application in regenerative medicine. Int Rev Cell Mol Biol 2014; 313:27-77). In addition, polyP induces the ALP (Müller W E G, Wang X H, Diehl-Seifert B, Kropf K, Schloßmacher U, Lieberwirth I, Glasser G, Wiens M and Schröder H C. Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Ca2+ level in osteoblasts (SaOS-2 cells) in vitro. Acta Biomater 2011; 7:2661-2671).
Bone Graft Materials
Bone graft materials must be biocompatible and comprise similar biomechanical properties like the physiological bone tissue. Favored are substitutes that are degradable and allow the surrounding tissue to migrate into at least the surface region of the non-self implant to strengthen the bridging of the biofabricated material to the surrounding tissue and to avoid inflammatory reactions.
Besides of cell-based bone graft substitutes ceramic-based materials have proven to be useful bone scaffold for implants. Among the calcium phosphate-based ceramics, the following materials are of particular relevance:
Hydroxyapatite (HA).
HA is produced at a high-temperature reaction and comprises a crystalline form of calcium phosphate (Noshi T, Yoshikawa T, Ikeuchi M, Dohi Y, Ohgushi H, Horiuchi K, Sugimura M, Ichijima K, Yonemasu K. Enhancement of the in vivo osteogenic potential of marrow/hydroxyapatite composites by bovine bone morphogenetic protein. J Biomed Mater Res 2000; 52:621-630). Since the composition of HA is Ca10(PO4)6(OH)2 with a calcium-to-phosphate ratio of 1.67 this material is chemically very similar to the mineralized phase of physiological bone. Based on this property HA shows suitable biocompatibility (Nandi S K, Kundu B, Ghosh S K, De D K, Basu D. Efficacy of nano-hydroxyapatite prepared by an aqueous solution combustion technique in healing bone defects of goat. J Vet Sci 2008; 9:183-191).
β-Tricalcium Phosphate (β-TCP).
Similar to HA, the chemical composition Ca3(PO4)2 and crystallinity of β-TCP match the ones of the mineral phase of bone; in addition, this material is bioabsorbable and biocompatible (Daculsi G, LeGeros R Z, Heughebaert M, Barbieux I. Formation of carbonate apatite crystals after implantation of calcium phosphate ceramics. Calcif Tissue Int 1990; 46:20-27). TCP implants have been successfully used as synthetic bone void fillers both in orthopedics and in dentistry (Shigaku S, Katsuyuki F. Beta-tricalcium phosphate as a bone graft substitute. Jikeikai Med J 2005; 52:47-54).
Bioactive Glass.
Finally, bioactive glass ceramics (“bioglass”) initially developed by Hench et al (Hench L L, Splinter R J, Allen W C, Greenlee T K. Bonding mechanisms at the interface of ceramic prosthetic materials. J Biomed Mater Res Symp 1971; 2:117-141; reviewed in: Chen Q, Roether J A, Boccaccini A R. Tissue engineering scaffolds from bioactive glass and composite materials. In: Ashammakhi N, Reis R, Chiellini F Topics in Tissue Engineering, Vol 4, pp. 1-27, 2008) is not only biocompatible but also osteoconductive and allows bone to bind without any intervening fibrous connective tissue interface (Zhang H, Ye X J, Li J S. Preparation and biocompatibility evaluation of apatite/wollastonite-derived porous bioactive glass ceramic scaffolds. Biomed Mater 2009; 4: 45007). This mineralic glass is frequently used as filling material for bone defects either alone or in combination with autogeneic or allogenic cancellous bone graft (Dorea H C, McLaughlin R M, Cantwell H D, Read R, Armbrust L, Pool R, Roush J K, Boyle C. Evaluation of healing in feline femoral defects filled with cancellous autograft, cancellous allograft or Bioglass. Vet Comp Orthop Traumatol 2005; 18:157-168) or with morphogenetically active inorganic polymers, e.g. polyphosphate [polyp] (Wang X H, Tolba E, Schröder H C, Neufurth M, Feng Q, Diehl-Seifert B, Müller W E G. Effect of bioglass on growth and biomineralization of SaOS-2 cells in hydrogel after 3D cell bioprinting. PLoS ONE 2014; 9:e112497).
Since all of these mineralic implant materials are fabricated at temperatures higher than 700° C. and, in turn, are not, or only at a limited degree, osteoinductive like the bioglass, the inventors developed a new potential biomaterial likewise suitable to be used as bone implant.
Skin Aging and Collagen
The epidermis of human skin is composed of keratinocytes of different proliferation and differentiation state, while the dermis is composed of three major types of cells, the fibroblasts, macrophages, and adipocytes. The most abundant protein in the skin and connective tissue, collagen type I, and the other fibrillar collagens, types III and V, are secreted as procollagens and then enzymatically processed to assemble to the triple helix configuration. The quantity and quality, integrity and biomechanical properties, of collagen decrease during skin aging, often accelerated by external factors (Pandel R, Poljšak B, Godic A, Dahmane R (2013) Skin photoaging and the role of antioxidants in its prevention. ISRN Dermatol 12; 2013:930164). Skin aging can be grouped into: i) Chronological or intrinsic aging, and ii) Solar aging (photoaging). Focusing on the photoaging process, it is well established that the aging skin is characterized by reduced amounts of collagen, accumulation of abnormal elastic fibers and, in parallel, increased quantities of glycosaminoglycans in the upper and mid dermis. The major reason for this imbalance in the extracellular matrix fibrillar meshwork is the occurrence of oxygen-derived species including free radicals (Gilchrest B A, Bohr V A. Aging process, DNA damage, and repair. FASEB J 1997; 11:322-330).
While the synthesis of collagen I remains almost unchanged in human skin during lifetime, the extent of collagen III formation drastically drops by 70%. The differential regulation of collagen gene expression type I versus type III is understood to some extent. Due to the exceptionally long half-life of collagen, the fibrils undergo nonenzymatic glycation under formation of advanced glycation end products (AGEs), through which several signaling pathways and collagen types I and III gene expression become modulated (Tang M, Zhong M, Shang Y, Lin H, Deng J, Jiang H, Lu H, Zhang Y, Zhang W. Differential regulation of collagen types I and III expression in cardiac fibroblasts by AGEs through TRB3/MAPK signaling pathway. Cell Mol Life Sci 2008; 65:2924-2932).
Retinoids are structurally characterized by a β-ionone ring with a polyunsaturated side chain consisting of four isoprenoid moieties with a terminal alcohol, aldehyde, carboxylic acid or ester group. It is well established that retinoids are beneficial for skin regeneration, reconstitution of the collagen network, and protective against skin aging (reviewed in: Mukherjee S, Date A, Patravale V, Korting H C, Roeder A, Weindl G. Retinoids in the treatment of skin aging: an overview of clinical efficacy and safety. Clin Intery Aging 2006; 1:327-348). In addition, retinol has been proposed to function as a stabilizer for biologically active skin preservatives (Nystrand G, Debois J. Retinol stabilized cleansing compositions; EP 995433 A1).
The application of polyP as a skin preservative is hampered by the fact that the salt Na-polyP is readily dissolved. The approaches to fabricate polyP-containing biomaterials that are more resistant and simultaneously bioactive included a calcination process. However, during those processes polyP is either disintegrated or transformed into a crystalline state and by that becomes inactive (Pilliar R M, Filiaggi M J, Wells J D, Grynpas M D, Kandel R A. Porous calcium polyphosphate scaffolds for bone substitute applications—in vitro characterization. Biomaterials 2001; 22:963-972; Ding Y L, Chen Y W, Qin Y J, Shi G Q, Yu X X, Wan C X. Effect of polymerization degree of calcium polyphosphate on its microstructure and in vitro degradation performance. J Mater Sci Mater Med 2008; 19:1291-1295).
The new materials according to this invention are based on polyP. Previously polyP has also been used, after calcinations, as potential scaffold for bone implants (Pilliar R M, Filiaggi M J, Wells J D, Grynpas M D, Kandel R A. Porous calcium polyphosphate scaffolds for bone substitute applications—in vitro characterization. Biomaterials 2001; 22:963-972; Qiu K, Wan C X, Zhao C S, Chen X, Tang C W, Chen Y W. Fabrication and characterization of porous calcium polyphosphate scaffolds. J Materials Sci 2006; 41:2429-2434; Ding Y L, Chen Y W, Qin Y J, Shi G Q, Yu X X, Wan C X. Effect of polymerization degree of calcium polyphosphate on its microstructure and in vitro degradation performance. J Mater Sci Mater Med 2008; 19:1291-1295; Wang D, Wallace A F, De Yoreo J J, Dove P M. Carboxylated molecules regulate magnesium content of amorphous calcium carbonates during calcification. Proc Natl Acad Sci USA 2009; 106:21511-21516). In all those preparations, polyP had to be subjected to calcination with the result that the polyP chain might be degraded and the exact chain length is impossible to be determined. Furthermore those polyP scaffolds have not been described to act in an osteoinductive manner.
The material according to this invention is a hard amorphous polyP-based biomaterial that is produced at ambient conditions (i.e. at around 20° C.±10° C.) in the presence of a distinctly adjusted concentration of CaCl2. The material obtained comprises a porous scaffold built of spherical, amorphous nanoparticles that are biodegradable and retain the morphogenetic activity of the inorganic polymer.
Thus, in a first aspect thereof, this invention concerns a new morphogenetically active material consisting of calcium polyphosphate (polyP) nanoparticles that are (i) amorphous and (ii) display an unusual hardness not found for calcium polyphosphate materials prepared by state-of-the-art methods. The inventors developed a controlled and slow fabrication process that is performed at room temperature and unexpectedly resulted in the formation of a material showing these properties. The polyP material formed in the presence of CaCl2 at a stoichiometric ratio of around 1 or 2 (phosphate to calcium) is an amorphous powder that is composed of nanospheres with a diameter of approximately between about 45 nm to about 0.25 μm. The inventive material is degradable, in contrast to the Ca-polyP salt prepared by conventional methods which resists hydrolytic cleavage by phosphatases present in medium over longer time periods in cell culture experiments. The polyP nanoparticles that form this nanoparticulate material are termed amorphous Ca-phosphate nanoparticles [aCa-polyP-NP].
The inventors further succeeded to include retinol into these nanoparticles and thereby to fabricate nanospheres, consisting of retinol inclusions encapsulated within Ca-polyP shells. These nanospheres are termed amorphous Ca-polyP/retinol nanospheres [retinol/aCa-polyP-NS]. The inventors unexpectedly found that this new, inventive material, the retinol/aCa-polyP-NS, causes collagen type III expression in an unexpected high extent, at concentrations at which the single components, retinol and aCa-polyP-NP, are biologically inactive.
The following patent applications on polyP are deemed relevant; GB1406840.7. Morphogenetically active hydrogel for bioprinting of bioartificial tissue. Inventors: Müller W E G, Schröder H C, Wang X H; GB1403899.6. Synergistic composition comprising quercetin and polyphosphate for treatment of bone disorders. Inventors: Müller W E G, Schröder H C, Wang X H.
In a first aspect of the invention, a novel polyphosphate (polyP) material is provided that is characterized by the following properties: a) The material is amorphous (non-crystalline); b) The material has an unusual hardness (e.g., the elastic modulus of the Ca-polyP2 biopolymer according to this invention amounts to approximately 1.3 GPa (in the context of the present invention, “hard” or “hardness” means a value of approximately 1.3 GPa, such as between 0.8 and 1.8 GPa, preferably between 1.0 and 1.6 GPa), close to value measured for trabecular tissue surrounding human bone [6.9 GPa]); and preferably, c) The material consist of nanoparticles with a diameter of about 45 nm to about 0.25 μm (Ca-polyP2 particles according to this invention). Advantageously, the inventive material can be prepared under mild conditions, such as ambient conditions, and particularly at room temperature.
Hardness can be measured using the Brinell and/or Vickers hardness test method, as known to the person of skill.
It was surprisingly found, that the inventive material is amorphous (non-crystalline), morphogenetically active, it induces bone alkaline phosphatase activity and new bone formation (hydroxyapatite synthesis); and that the material is biodegradable (e.g. by polyphosphatases, such as bone alkaline phosphatase).
This inventive material, whose properties make it superior compared to conventional polyphosphate preparations for a use, for example, in bone regeneration and replacement materials (e.g. GB1406840.7. Morphogenetically active hydrogel for bioprinting of bioartificial tissue [Inventors: Müller W E G, Schröder H C, Wang X H]; GB1403899.6. Synergistic composition comprising quercetin and polyphosphate for treatment of bone disorders [Inventors: Müller W E G, Schröder H C, Wang X H]), can be prepared according to the following method, according to this invention:
a) Dissolution of a polyP salt in water and adjustment of the pH to alkaline values,
b) (slow) Addition of a solution of a calcium salt to the polyP salt (with Na+) solution, and adjustment of the pH to alkaline value, and
c) Collection and drying of the particles thus formed, optionally after washing with ethanol.
This procedure is performed at room temperature.
In another aspect, this invention relates to a method for the fabrication of nanospheres that are composed of (i) Ca2+ together with (ii) polyP to form nanoparticles, aCa-polyP-NP, and together with (iii) retinol that is encapsulated by those nanoparticles to form nanospheres.
Unexpectedly, the inventors found that processing of these nanoparticles with retinol and poly(ethylene glycol) [PEG] results in the formation of nanospheres, retinol/aCa-polyP-NS, which show the following unexpected and advantageous properties:
1. The nanospheres according to this invention, retinol/aCa-polyP-NS, are highly homogenous in size (size ˜45 nm). This size is optimal for endocytotic cellular uptake (Zhang S, Li J, Lykotrafitis G, Bao G, Suresh S. Size-dependent endocytosis of nanoparticles. Adv Mater 2009; 21:419-424).2. In contrast, the nanoparticles, aCa-polyP-NP, are large (>50 μm sized) brick-like particles.3. Both components of the nanospheres, retinol and polyP, act synergistically: if given together at “non-effective” concentrations, a strong increase in proliferation of cells occurs.4. Both components of the nanospheres, retinol and polyP, cause a highly synergistic effect on the expression of collagen types I and II, and especially collagen type III. The effects are already observed at concentrations of retinol and aCa-polyP-NP that do not display any biological effect if administered alone. Even though the application of retinol at concentrations of ≤1% (36 mM) is assessed as a safe cosmetic ingredient higher concentrations might display adverse effects, e.g. inhibition of responses to viral or chemical carcinogens (see: Final report on the safety assessment of retinol palmitate and retinol. J Americ Coll Toxicol 1987; 6:279-319). Therefore, the nanospheres according to the invention open a more safe application of this compound, e.g. in cosmetics.
The nanospheres according to this invention can be disintegrated by enzymatic hydrolysis in the extracellular space. The main component within the nanospheres, polyP, undergoes degradation by the exo-phosphatase, most likely by the alkaline phosphatase which is involved in the extracellular degradation of polyP (Müller W E G, Wang X H, Diehl-Seifert B, Kropf K, Schloßmacher U, Lieberwirth I, Glasser G, Wiens M, Schröder H C. Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Ca2+ level in osteoblasts (SaOS-2 cells) in vitro. Acta Biomaterialia 2011j; 7:2661-2671). In consequence, not only polyP is released extracellularly but also retinol.
The nanospheres according to this aspect of the invention have the appropriate size to be taken up by a clathrin-mediated endocytosis process. Their biological activity can be blocked with the endocytosis inhibitor triflupromazine. This property of the nanospheres is advantageous under conditions during which the transmembrane retinol transporter is down-regulated (resulting in epidermal thickening).
In consequence, the nanospheres show a dual biological effect of the two active components, polyP and retinol—both via the extracellular route (activation of cell membrane-bound receptors) and via the transmembrane/intracellular route (through endocytosis).
The nanospheres according to this invention are not cytotoxic.
As a preferred example, the method can be carried out using sodium polyphosphate (Na-polyP) as a polyP salt (chain length: 50 phosphate units) in solution and calcium chloride as a calcium salt in solution as follows.
a) Dissolution of 10 g of Na-polyP in 500 ml of distilled water and adjustment of the pH to 10 with 1 M NaOH (room temperature),
b) Slow, dropwise (1 ml/min), addition of a solution of 14 g of calcium chloride (Ca-polyP1) or 28 g of calcium chloride (Ca-polyP2) in 250 ml salt to the Na-polyP solution and adjustment of the pH to 10 (room temperature),
c) Stirring of the thus formed suspension for about 4 h, and
d) Collection of the particles formed, and optionally washing them with ethanol while filtering (0.45 μm filter; e.g., Nalgene Rapid-Flow), and drying at 60° C.
The Ca-polyP material obtained with 14 g of CaCl2 was named Ca-polyP1 (stoichiometric ratio phosphate:calcium of 1); and the Ca-polyP material obtained with 28 g of CaCl2 was named “Ca-polyP2” (stoichiometric ratio phosphate:calcium of 1:2).
The inventive material, consisting of amorphous retinoid/Ca-polyP nanospheres, which are superior compared to the individual (retinoid and polyP) components, can be prepared according to this invention as follows: a) Dissolution of a retinoid and a calcium salt in an organic solvent, b) Slow addition of the retinoid calcium salt solution to an aqueous polyP solution, and c) Collection and drying of the nanospheres formed after washing with water. This procedure is performed at room temperature, under avoidance of light.
As an example, the preparation of the amorphous retinol/Ca-polyP nanospheres (retinol/aCa-polyP-NS) can be carried out using Na-polyP as a polyP salt (chain length: 30 phosphate units) and calcium chloride as a calcium salt as follows. A) Preparation of a solution containing 100 mg of retinol and 2.8 g of CaCl2 in 50 ml absolute ethanol (=Retinol/calcium solution). B) Preparation of a polyP solution containing 1 g of polyP in 100 ml water; in order to avoid a phase separation and to stabilize the emulsion, 2 g of poly(ethylene glycol) [PEG] are added to the Na-polyP solution (=PolyP solution). C) Drop-wise addition of the Retinol calcium solution to the PolyP solution. D) Stirring of the emulsion formed for 6 h. E) Collection of the nanospheres formed by filtration and washing with water to remove excess of calcium ions and unreacted components. F) Drying the particles at room temperature overnight.
The nanoparticles comprising or consisting of the inventive Ca-polyP material are characterized by a high hardness (about 1.3 GPa; Ca-polyP2) compared to larger particles that are produced at a different phosphate to calcium ratio in solution or at harsh reaction conditions, e.g. in acid-flux of phosphoric acid with Ca(OH)2 at high (250° C.) temperature (Jackson L E, Kariuki B M, Smith M E, Barralet J E, Wright A J. Synthesis and structure of a calcium polyphosphate with a unique criss-cross arrangement of helical phosphate chains. Chem Mater 2005; 17:4642-4646), or the described calcium-polyphosphate complex (Müller W E G, Wang X H, Diehl-Seifert B, Kropf K, Schloßmacher U, Lieberwirth I, Glasser G, Wiens M, Schröder H C. Inorganic polymeric phosphate/polyphosphate is an inducer of alkaline phosphatase and a modulator of intracellular Ca2+ level in osteoblasts (SaOS-2 cells) in vitro. Acta Biomater 2011; 7:2661-2671).
The new and hard polyP material according to this invention is biodegradable and displays superior morphogenetic activity, compared to the Ca-polyP salts prepared by conventional techniques.
In addition, the inventive hard Ca-polyP nanoparticles are prone to cellular uptake (even observed for larger, 600 nm particles; Zhao Y, Sun X, Zhang G, Trewyn B G, Slowing I I, Lin V S. Interaction of mesoporous silica nanoparticles with human red blood cell membranes: size and surface effects. ACS Nano 2011; 5:1366-1375) and subsequent metabolization, a property that is not possible for the free polyanionic polyP polymer.
The chain lengths of the polyP molecules can be in the range 3 to up to 1000 phosphate units. Optimal results are achieved with polyP molecules with an average chain length of approximately 200 to 20, and optimally about 30 to 50 phosphate units.
A further aspect of the invention concerns the material as obtained by one of the methods described above.
Further, the inventors demonstrate that the inventive method can be used for the preparation of hard amorphous and morphogenetically active polyP nanoparticles.
A further aspect of the invention concerns a material comprising the nanoparticles as obtained by one of the methods described above.
The technology according to this invention can be used for the fabrication of nanoparticles or for a material containing such nanoparticles to be used, preferably, in bone regeneration and repair and in dentistry.
A further aspect of the invention concerns the application of the inventive nanoparticles in drug delivery, again, preferably, in bone regeneration and repair and in dentistry, in analogy to, for example, systems described in Kwon et al. and Yang et al. (Kwon S, Singh R K, Perez R A, Abou Neel E A, Kim H W, Chrzanowski W. Silica-based mesoporous nanoparticles for controlled drug delivery. J Tissue Eng. 2013 Sep. 3; 4:2041731413503357. eCollection 2013. Yang P, Gai S, Lin J. Functionalized mesoporous silica materials for controlled drug delivery. Chem Soc Rev. 2012 May 7; 41(9):3679-98).
A further aspect of the invention concerns a material such as a crème or ointment containing such retinol/calcium-polyphosphate nanospheres (retinol/aCa-polyP-NS) obtained by one of the methods described above.
The technology according to this invention can be applied for the fabrication of nanospheres or a material containing these nanospheres, such as a crème or ointment, to be used in the treatment or prophylaxis of dermatological conditions such as inflammatory skin disorders, acne, disorders of increased cell turnover like psoriasis, skin cancers, and photoaging.
A further aspect of the invention concerns the application of the inventive nanospheres in drug delivery.
The invention will now be described further in the following preferred examples, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties.